Rationally Designed Solvatochromic ... - Wiley Online Library

DOI: 10.1002/chem.201701425

Communication

& Solvatochromism

Rationally Designed Solvatochromic Fluorescent Indoline Derivatives for Probing Mitochondrial Environment Kaushik Pal and Apurba Lal Koner*[a] search for new small, robust, and bright fluorescent reporters with a suitable functional group for specific functionalization or targeting the organelle of interest with good photochemical stability, combined with low cellular cytotoxicity.[4e, 10] Moreover, water solubility, pH stability, and indifference of the emissive properties of the reporter fluorescent molecule towards ionic strength are equally important for cellular imaging.[8c, 11] To develop a water-soluble fluorescent dye with the specific functional group, desirable reporter properties such as brightness or photo- and thermal stability are often sacrificed.[12] Eventually, the objective is to achieve specific targeting of a BOI or cel-

Abstract: A new class of solvatochromic, robust, and multifunctional fluorescent probes derived from indoline is presented. Specificity of mitochondria targeting was achieved and utilized for probing polarity under normal and apoptotic conditions. A large Stokes shift, high quantum yield, thermal, photochemical, and pH stability, tolerance to buffer compositions, and a bioconjugation tool-kit make it a promising candidate for live-cell fluorescence imaging.

As the powerhouse of the cell, mitochondria play a vital role in maintaining regulated cellular processes such as redox stress, pyruvate and citrate cycle, and storing of metal ions.[1] The results of regulated physiochemical reactions are strongly controlled by the chemical and physical environment.[2] Consequently, the investigation of the physical and chemical environment inside mitochondria is one of the most fundamental quests and demanding tasks for modern researchers.[3] Additionally, selective targeting of a biomolecule of interest (BOI) among a myriad of different functional molecules to unravel its spatiotemporal dynamics is also an emerging and challenging area in biophysical research.[4] In this regard, fluorescence microscopy is one of the most widely used techniques.[5] Most BOIs are intrinsically either non-fluorescent or too weakly fluorescent to visualize them over an auto-fluorescent background.[5b] Moreover, externally incorporated fluorophores are devoid of good water-solubility, environmental sensitivity, and specific targeting abilities for desired optical properties in physiological conditions. Fortunately, with the help of organic synthetic and chemical biology tools, one can visualize BOIs by tagging a suitable fluorescent molecule.[6] Fluorescent organic dyes, quantum dots (QDs), metal clusters, and polymer dots are commonly used as reporters for fluorescence imaging.[7] Generally, fluorescent dyes are inferior owing to their poor photostability.[8] In comparison, QDs are highly photostable but their usability is limited owing to their larger size and longterm heavy-metal toxicity.[9] Therefore, there is a constant [a] K. Pal, Dr. A. L. Koner Department of Chemistry Indian Institution of Science Education and Research Bhopal Bhopal Bypass Road, Bhauri, Bhopal-462066 (India) E-mail: [email protected] Supporting Information and the ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/ chem.201701425. Chem. Eur. J. 2017, 23, 8610 – 8614

Figure 1. (a) Multifunctional Propellerocein dye shows structural resemblance with a propeller; (b) synthesis of Propellerocein derivatives.

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Communication lular organelle with a reporter fluorophore comprising all desirable properties to visualize its dynamics in the cellular environment or determine the local chemical and physical status.[13] With the advancement of super-resolution imaging, the design and synthesis of new fluorescent probes with all prerequisites remains an open quest.[14] Herein, we have reported the design and synthesis of a new class of 2,3-diketoindoline-based propeller-shaped (denoted as Propellerocein) multifunctional fluorescent dyes with high tolerance to buffer solutions and the whole pH range along with high photo- and thermal stability. Bioconjugation efficiency of the probe was validated by chemical ligation with Traptavidin (Tr) and thiol-functionalized DNA. Furthermore, the mitochondria-targeting derivative 15 was utilized for specific targeting of mitochondria for the micropolarity determination under normal and apoptotic conditions. A poor yield (< 1 %) of the desired fluorescent product limits its applications.[15] Recently, the synthesis and photophysical properties of 1,2-dihydroindol-3-one derivatives with expanded conjugation have been reported.[16] We designed and synthesized the multifunctional fluorescent probe for bioconjugation and deciphering the microenvironment inside living systems. The single-crystal X-ray structure of compound 7 confirms the propeller-like shape of the fluorescent core (Figure 1 a). Optimization of the Grignard reaction conditions resulted in 24 % yield of the desired product (Figure 1 b, see the Supporting Information for a detailed characterization). An n-propyl chain appended with a hydroxyl group was anchored to the fluorescent core to avoid steric hindrance from bulky BOIs. This incor-

porated hydroxyl group was further modified to other functional groups for selective bioconjugation. For specific bioconjugation and organelle targeting seven different derivatives [hydroxyl (5) and amine (8) to target carboxylic acids or NHSesters, alkyne (9) and azide (7) for bio-orthogonal click chemistry, maleimide (11) specific for thiols, NHS-ester (13) for free amine groups, and triphenylphosphonium cation (15) to specifically target mitochondria] were synthesized by using CuIcatalyzed alkyne–azide click chemistry. The specifically fused entity made of a BOI and a fluorescent probe needs to be characterized by spectrophotometry, fluorescence spectroscopy, and microscopy. Propellerocein contains the indoline N, which is capable of easily donating its electron lone pair to the electron-deficient benzoyl ketone part through the phenyl ring (Figure S1a in the Supporting Information). A molecule containing both electron-rich and -deficient groups connected with a p-conjugated linker is generally known as an intramolecular charge-transfer (ICT) dye.[17] Facile charge transfer makes ICT dyes highly dipolar both in the ground and electronically excited state. Propellerocein 5 was selected for photophysical studies because all other dyes were obtained by its modification at the remote position. Solvent polarity-dependent UV/Vis and fluorescence measurements showed the classical ICT-dye character of 5. The study showed a small shift in the absorption maxima with the solvent polarity (Table S1 and Figure S1b in the Supporting Information). In contrast, the emission maxima shifted towards longer wavelengths with the increase of polarity (Figure 2 a, Table S1 and Figure S2 in the Supporting Information),

Figure 2. Solvent-dependent fluorescence properties of Propellerocein 5: (a) normalized fluorescence spectra with varying polarity of the solvent, shows redshifted emission maxima in the polar solvent; (b) Stokes shift of the probe varies linearly (R2 = 0.93) with the solvent polarity according to ET(30) scale; (c) time-dependent fluorescence-decay profile of 5 in solvents with different polarity, exhibiting longer fluorescence lifetime in the polar solvent; (d) fluorescence lifetime of the probe varies linearly (R2 = 0.89) according to ET(30) scale. Chem. Eur. J. 2017, 23, 8610 – 8614

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Communication which confirmed the ICT-dye properties. An excellent overlap between absorption and excitation spectra of 5 in water confirmed its optical purity (Figure S3 in the Supporting Information). The Stokes shift and emission maxima increased linearly with the solvent polarity scale ET(30) (Figure 2 b and Figure S4 in the Supporting Information).[18] A large Stokes shift and high fluorescence quantum yield (Table S1 in the Supporting Information) were expected owing to the non-twisted ICT character and make 5 a potent probe for fluorescence imaging. To gain further insight into the photophysical properties, we also performed time-resolved fluorescence experiments. Time-correlated single-photon counting (TCSPC) results and steady-state data showed excellent agreement (Figure 2 c). The probe exhibited very long excited-state lifetime, which increased linearly with the solvent polarity (Figure 2 d and Table S1 in the Supporting Information). This result also opens up an opportunity to use this probe for fluorescence lifetime imaging. Photo-bleaching of fluorescent reporters limits monitoring of any time-dependent biological events owing to long-time exposure to excitation light. Similarly, fluorescence quenching can also occur owing to local heating of the sample. Therefore, for long-term fluorescence imaging the photochemical and thermal stability of the reporter fluorescent probe are prerequi-

sites. Propellerocein showed six times higher photochemical stability in phosphate buffer solution (PBS) compared to Fluorescein (Figure 3 a, Figure S5 in the Supporting Information).[19] After 90 min illumination, only approximately 2–7 % reduction in the fluorescence intensity of 5 was observed (inset Figure 3 b) compared to 30–35 % in the case of Fluorescein. Owing to the local heating originating from the intense light source it is always advisable to use a fluorescent probe with good thermal stability.[20] We examined the thermal stability of the present probe from 20 to 70 8C in PBS buffer, for which its fluorescence intensity remained unchanged (Figure S6 in the Supporting Information). A perfect fluorescent probe attached to a BOI should remain unaltered in terms of emission behavior when subjected to variable environments because the biological system is a realm of heterogeneity in terms of pH, viscosity, ionic strength, etc. The photophysical properties of 5 were almost indifferent to various pH values (Figure 3 c). Such pH resilience can be rationalized owing to the absence of any ionizable functional groups that could be affected in the studied pH window. To mimic the physiological or biological environment, biochemical experiments with live cells are usually performed in different buffers. However, very often fluorescence intensity of reporter probes

Figure 3. Photostability, an effect of different pH values and buffer solutions on fluorescence properties of 5: (a) photo-bleaching of 5 (black) and Fluorescein (red) followed through the decrease of the absorption with increasing time under white-light irradiation in PBS (pH 7.5); (b) reduction of fluorescence intensity upon continuous illumination; the inset shows the relative reduction of the fluorescence intensity of the probe and Fluorescein at 515 nm against irradiation time; (c) fluorescence intensity of 5 at 515 nm upon excitation at 440 nm plotted against the whole scale of pH; (d) fluorescence intensity of 5 at 515 nm upon excitation at 440 nm in commonly used buffer and other solutions, shows retention of emission intensity irrespective of the composition; standard deviation is from triplicated measurements. Chem. Eur. J. 2017, 23, 8610 – 8614

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Communication tagged with BOIs is quenched significantly in different buffer solutions owing to the presence of added supplements, which destroys the utility of those tags. In this regard, we investigated the fluorescence behavior of 5 in commonly used buffer solutions. Along with those relevant buffer media, we also tested the effect of 10 % SDS–PAGE running medium, and 0.1 m imidazole in PBS. The probe showed excellent retention of fluorescence intensity in each tested buffer or medium (Figure 3 d). Chemical fusion of biomolecules with a suitable fluorescent reporter is a common and useful practice in chemical biology.[21] After the critical assessment, we further explored the potential of Propellerocein derivatives for bioconjugation and live-cell imaging beause they have minimal cytotoxicty (Figure S8 in the Supporting Information). The scope of covalent functionalization using the NHS derivative 13 was extended by covalent conjugation with Tr. Firstly, we conjugated the amine groups on Tr by using the NHS ester derivative 13 and quantified the average dye number per Tr (for details, see the Supporting Information). To investigate the biotin binding ability of purified 13-Tr conjugate, it was further tested using SDSPAGE protein gel through a mobility-shift assay (Figure S50a, c in the Supporting Information). Further, we selected the maleimide 11 for thiolated DNA conjugation. A thiolated 439-bp DNA was generated by using polymerase chain reaction (PCR). Thiol-modified DNA was incubated with 11 in PBS (pH 7.5), and the final product was tested in the agarose gel (Figure S50b, d in the Supporting Information). The large negative membrane potential (DY = @150 to @180 mV) of mitochondria makes probe 15 potent for the specific staining of mitochondria containing the triphenylphosphonium cation, as indicated in previous reports.[22] The optical properties of 15 are very similar to its parent precursor 5 in water (Table S2 in the Supporting Information). Firstly, we performed co-localization fluorescence microscopy with 15 and commercially available Mito-Tracker red by using a confocal microscope (Figure 4 a–d, Figure S9 in the Supporting Information) with the live breast cancer cell line MDA-MB-231. The experiment revealed that probe 15 specifically localized mitochondria with a high Pearson’s coefficient of approximately 0.93 : 0.02 (Figure 4 e). Because Propellerocein 15 can specifically accumulate inside mitochondria and its fluorescence maximum follows a linear relationship with local polarity, it is a perfect candidate to determine the polarity inside mitochondria under various physiological conditions. We used spectral scanning and linear unmixing techniques (see the Supporting Information) in a confocal microscope, during which the microscope was used as a spectrometer. The emission maximum was obtained around 482 nm (Figure 4 f and Figure S52 in the Supporting Information), resembling a mixture of acetonitrile and isopropanol. During apoptosis, cells undergo a tremendous amount of biochemical changes, and the outcomes of such biochemical pathways are strongly related to the mitochondrial environment. We determined the modulation of micro-polarity inside mitochondria under peroxide-induced (1 mm H2O2) apoptotic conditions. From this experiment, we obtained an average Chem. Eur. J. 2017, 23, 8610 – 8614

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Figure 4. Fluorescence colocalization microscopy and spectroscopy inside the live cell MDA-MB-231 with 15 and Mito-Tracker red: (a) incubated with 1 mm 15, collected in green channel; (b) incubated with 0.3 mm Mito-Tracker red, collected in red channel; (c) transmission differential interference contrast (DIC); (d) merging of all channels; (e) intensity correlation plot of 15 (horizontal axis) and Mito-Tracker red (vertical axis); Pearson’s coefficient 0.93 : 0.02; (f) emission spectral profile of 15 inside mitochondria, maximum at approximately 482 nm.

emission maximum of approximately 477 nm, which is in good agreement with our previous experiment performed with healthy cells. In contrast, we observed approximately 12 nm red-shift in the emission maxima of 15 inside mitochondria in apoptotic cells (Figure S53 in the Supporting Information). This indicates that during apoptosis the environment inside the mitochondria becomes much more polar compared to normal cellular conditions. This might be caused by the generation of a higher amount of reactive oxygen species during the apoptosis process. In conclusion, we have re-discovered 2,3-diketoindolinebased solvatochromic and multifunctional fluorescent probes through design and synthesis. In addition to their multifunctionality, their small size, water-solubility, high brightness, large Stokes shift, high temperature tolerance, good photostability, and retention of fluorescence intensity in various buffers and pH values make them potent candidates for various biochemical applications. Furthermore, their specific bioconjugation ability with proteins and DNA was tested. Finally, a mitochon-

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Communication dria-specific derivative was utilized to determine the polarity inside mitochondria of healthy and apoptotic cells. Hence, we believe that this new class of Propellerocein probes can serve as a one-step solution for fluorescence imaging and singlemolecule spectroscopy inside a specific organelle of a live cell.

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[8]

Acknowledgements We would like to thank IISER Bhopal, India for generous startup funding. K.P. gratefully acknowledges Council of Scientific & Industrial Research, India for the doctoral fellowship.

Conflict of interest

[9]

[10]

[11]

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Manuscript received: March 30, 2017 Accepted manuscript online: May 4, 2017 Version of record online: June 5, 2017

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